Modification of Arg-13 of mu-conotoxin GIIIA with piperidinyl-Arg analogs and their relation to the inhibition of sodium channels

mu-Conotoxin GIIIA, a peptide toxin isolated from the marine snail Conus geographus, preferentially blocks skeletal muscle sodium channels in vertebrates. In this study, analogs of mu-conotoxin GIIIA in which essential Arg-13 was replaced with arginine analogs consisting of a piperidyl framework to regulate length and direction of the side chain were synthesized. Synthesized analogs exhibited similar CD and NMR spectra to that of GIIIA, suggesting a three-dimensional structure identical to that of the native toxin. The biological activities of piperidyl analogs were decreased or lost despite the small change in the side chain of Arg-13. The investigated structure-activity relationships in inhibiting electrically stimulated muscle contraction suggest that the guanidinium group at amino acid position 13 interacts best when spaced with three to four carbons and placed in a vertical direction from the peptide loop. Thus, the position of the guanidinium group at Arg-13 of GIIIA must be located in a certain range for its strong interaction with the channel protein.     

mu-conotoxin GIIIA, a peptide ligand for muscle sodium channels: chemical synthesis, radiolabeling, and receptor characterization

The peptide conotoxin GIIIA from Conus geographus L. venom, which specifically blocks sodium channels in muscle, has been synthesized by a solid-phase method. The three disulfide bridges were formed by air oxidation. After HPLC purification, the synthetic product was shown to be identical with the native conotoxin GIIIA from Conus geographus. A high specific activity, 125I derivative of mu-conotoxin was prepared and used for binding assays to the Na channel from Electrophorus electric organ. Specific binding could be abolished by competition with tetrodotoxin. The radiolabeled toxin was specifically cross-linked to the Na channel. These studies demonstrate that mu-conotoxin GIIIA can be used to define the guanidinium toxin binding site and will be a useful ligand for understanding functionally important differences between Na channel subtypes.     

Generation of polyclonal antibody against mu-conotoxin GIIIA using an immunogen of [Cys(5)]mu-conotoxin GIIIA site-specifically conjugated with bovine serum albumin.

mu-Conotoxin GIIIA, one of the strong peptide toxins in the cone shell, preferentially blocks the skeletal muscle-type sodium channels in vertebrates. The toxicity of mu-conotoxin GIIIA is nearly equal to that of tetrodotoxin. The generation of an antibody for the native toxins is analytically useful, but practically difficult due to its high toxicity to animals. In this study, we generated the polyclonal antibody for mu-conotoxin GIIIA using a specific conjugation method in which the immunogen was detoxified while retaining the active-site structure for the sodium channels. ELISA analysis showed that the generated antibody recognized the native toxin folded with three disulfide bridges, but not the linear one. Furthermore, the physiologically active mutants of GIIIA were recognized while the inactive mutants were not, suggesting that the newly generated antibody can selectively recognize the physiologically active toxins. These methods for generating an antibody against peptide toxins will be applicable to other peptide toxins.     

Multiple, distributed interactions of μ-conotoxin PIIIA associated with broad targeting among voltage-gated sodium channels

The first μ-conotoxin studied, μCTX GIIIA, preferentially blocked voltage-gated skeletal muscle sodium channels, Na(v)1.4, while μCTX PIIIA was the first to show significant blocking action against neuronal voltage-gated sodium channels. PIIIA shares >60% sequence identity with the well-studied GIIIA, and both toxins preferentially block the skeletal muscle sodium channel isoform. Two important features of blocking by wild-type GIIIA are the toxin's high binding affinity and the completeness of block of a single channel by a bound toxin molecule. With GIIIA, neutral replacement of the critical residue, Arg-13, allows a residual single-channel current (~30% of the unblocked, unitary amplitude) when the mutant toxin is bound to the channel and reduces the binding affinity of the toxin for Na(v)1.4 (~100-fold) [Becker, S., et al. (1992) Biochemistry 31, 8229-8238]. The homologous residue in PIIIA, Arg-14, is also essential for completeness of block but less important in the toxin's binding affinity (~55% residual current and ~11-fold decrease in affinity when substituted with alanine or glutamine). The weakened dominance of this key arginine in PIIIA is also seen in the fact that there is not just one (R13 in GIIIA) but three basic residues (R12, R14, and K17) for which individual neutral replacement enables a substantial residual current through the bound channel. We suggest that, despite a high degree of sequence conservation between GIIIA and PIIIA, the weaker dependence of PIIIA's action on its key arginine and the presence of a nonconserved histidine near the C-terminus may contribute to the greater promiscuity of its interactions with different sodium channel isoforms.     

Docking of mu-conotoxin GIIIA in the sodium channel outer vestibule

mu-Conotoxin GIIIA (mu-CTX) is a high-affinity ligand for the outer vestibule of selected isoforms of the voltage-gated Na(+) channel. The detailed bases for the toxin's high affinity binding and isoform selectivity are unclear. The outer vestibule is lined by four pore-forming (P) loops, each with an acidic residue near the mouth of the vestibule. mu-CTX has seven positively charged residues that may interact with these acidic P-loop residues. Using pair-wise alanine replacement of charged toxin and channel residues, in conjunction with double mutant cycle analysis, we determined coupling energies for specific interactions between each P-loop acidic residue and selected toxin residues to systematically establish quantitative restraints on the toxin orientation in the outer vestibule. Xenopus oocytes were injected with the mutant or native Na(+) channel mRNA, and currents measured by two-electrode voltage clamp. Mutant cycle analysis revealed novel, strong, toxin-channel interactions between K9/E403, K11/D1241, K11/D1532, and R19/D1532. Experimentally determined coupling energies for interacting residue pairs provided restraints for molecular dynamics simulations of mu-CTX docking. Our simulations suggest a refined orientation of the toxin in the pore, with toxin basic side-chains playing key roles in high-affinity binding. This modeling also provides a set of testable predictions for toxin-channel interactions, hitherto not described, that may contribute to high-affinity binding and channel isoform selectivity.     

Synthesis of [Cys(5)]mu-conotoxin GIIIA and its derivatives as a probe of Na(+) channel analysis

The residue of Thr-5 in mu-conotoxin GIIIA (GIIIA), a receptor site I sodium channel blocker, was replaced with Cys. The synthesized [Cys(5)]GIIIA had a similar 3D structure to the native GIIIA, revealed by CD and NMR. [Cys(5)]GIIIA and its tagged peptides inhibited the electrically stimulated contraction of the rat diaphragm with relatively comparable potency to that of GIIIA. Since the contractile response to electrical stimuli is caused by the activation of sodium channels, [Cys(5)]GIIIA could be a prototype for synthesizing useful tools for the analysis of sodium channels. Thus, [Cys(5)]GIIIA could be a prototype for synthesizing useful tools for the analysis of sodium channels.     

Folding similarity of the outer pore region in prokaryotic and eukaryotic sodium channels revealed by docking of conotoxins GIIIA,PIIIA, and KIIIA in a NavAb-based model of Nav1.4

Voltage-gated sodium channels are targets for many drugs and toxins. However, the rational design of medically relevant channel modulators is hampered by the lack of x-ray structures of eukaryotic channels. Here, we used a homology model based on the x-ray structure of the NavAb prokaryotic sodium channel together with published experimental data to analyze interactions of the μ-conotoxins GIIIA, PIIIA, and KIIIA with the Nav1.4 eukaryotic channel. Using Monte Carlo energy minimizations and published experimentally defined pairwise contacts as distance constraints, we developed a model in which specific contacts between GIIIA and Nav1.4 were readily reproduced without deformation of the channel or toxin backbones. Computed energies of specific interactions between individual residues of GIIIA and the channel correlated with experimental estimates. The predicted complexes of PIIIA and KIIIA with Nav1.4 are consistent with a large body of experimental data. In particular, a model of Nav1.4 interactions with KIIIA and tetrodotoxin (TTX) indicated that TTX can pass between Nav1.4 and channel-bound KIIIA to reach its binding site at the selectivity filter. Our models also allowed us to explain experimental data that currently lack structural interpretations. For instance, consistent with the incomplete block observed with KIIIA and some GIIIA and PIIIA mutants, our computations predict an uninterrupted pathway for sodium ions between the extracellular space and the selectivity filter if at least one of the four outer carboxylates is not bound to the toxin. We found a good correlation between computational and experimental data on complete and incomplete channel block by native and mutant toxins. Thus, our study suggests similar folding of the outer pore region in eukaryotic and prokaryotic sodium channels.     

Reconstitution of the voltage-sensitive sodium channel of rat brain from solubilized components

The voltage-sensitive sodium channel of rat brain synaptosomes was solubilized with sodium cholate. The solubilized sodium channel migrated on a sucrose density gradient with an apparent S20,w of approximately 12 S, retained [3H]saxitoxin ([3H]STX) binding activity that was labile at 36 degrees C but no longer bound 125I-labeled scorpion toxin (125I-ScTX). Following reconstitution into phosphatidylcholine vesicles, the channel regained 125I-ScTX binding and thermal stability of [3H]STX binding. Approximately 50% of the [3H]STX binding activity and 58% of 125I-ScTX binding activity were recovered after reconstitution. The reconstituted sodium channel bound STX and ScTX with KD values of 5 and 10 nM, respectively. Under depolarized conditions, veratridine enhanced the binding of 125I-ScTX with a K0.5 of 20 microM. These KD and K0.5 values are similar to those of the native synaptosome sodium channel. 125I-ScTX binding to the reconstituted sodium channel, as with the native channel, was voltage dependent. The KD for 125I-ScTX increased with depolarization. This voltage dependence was used to demonstrate that the reconstituted channel transports Na+. Activation of sodium channels by veratridine under conditions expected to cause hyperpolarization of the reconstituted vesicles increased 125I-ScTX binding 3-fold. This increased binding was blocked by STX with K0.5 = 5 nM. These data indicate that reconstituted sodium channels can transport Na+ and hyperpolarize the reconstituted vesicles. Thus, incorporation of solubilized synaptosomal sodium channels into phosphatidylcholine vesicles results in recovery of toxin binding and action at each of the three neurotoxin receptor sites and restoration of Na+ transport by the reconstituted channels.     

Molecular Dynamics Study of Binding of μ-Conotoxin GIIIA to the Voltage-Gated Sodium Channel Nav1.4

Homology models of mammalian voltage-gated sodium (Na_V) channels based on the crystal structures of the bacterial counterparts are needed to interpret the functional data on sodium channels and understand how they operate. Such models would also be invaluable in structure-based design of therapeutics for diseases involving sodium channels such as chronic pain and heart diseases. Here we construct a homology model for the pore domain of the Na_V1.4 channel and use the functional data for the binding of µ-conotoxin GIIIA to Na_V1.4 to validate the model. The initial poses for the Na_V1.4–GIIIA complex are obtained using the HADDOCK protein docking program, which are then refined in molecular dynamics simulations. The binding mode for the final complex is shown to be in broad agreement with the available mutagenesis data. The standard binding free energy, determined from the potential of mean force calculations, is also in good agreement with the experimental value. Because the pore domains of Na_V1 channels are highly homologous, the model constructed for Na_V1.4 will provide an excellent template for other Na_V1 channels.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Structure-activity relationships of mu-conotoxin GIIIA: structure determination of active and inactive sodium channel blocker peptides by NMR and simulated annealing calculations.

A synthetic replacement study of the amino acid residues of mu-conotoxin GIIIA, a peptide blocker for muscle sodium channels, has recently shown that the conformation formed by three disulfide bridges and the molecular basicity, especially the one around the Arg13 residue, are important for blocking activity. In the present study, we determined the three-dimensional structure of an inactive analog, [Ala13]mu-conotoxin GIIIA, and refined that of the native toxin by NMR spectroscopy combined with simulated annealing calculations. The atomic root-mean-square difference of the mutant from the native conotoxin was 0.62 A for the backbone atoms (N, C alpha, C') of all residues except for the two terminal residues. The observation that the replacement of Arg13 by Ala13 does not significantly change the molecular conformation suggests that the loss of activity is not due to the conformational change but to the direct interaction of the essential Arg13 residue with the sodium channel molecules. In the determined structure, important residues for the activity, Arg13, Lys16, Hyp(hydroxyproline)17, and Arg19, are clustered on one side of the molecule, an observation which suggests that this face of the molecule associates with the receptor site of sodium channels. The hydroxyl group of Hyp17 is suggested to interact with the channel site with which the essential hydroxyl groups of tetrodotoxin and saxitoxin interact.     

Conus geographus toxins that discriminate between neuronal and muscle sodium channels

We describe the properties of a family of 22-amino acid peptides, the mu-conotoxins, which are useful probes for investigating voltage-dependent sodium channels of excitable tissues. The mu-conotoxins are present in the venom of the piscivorous marine snail, Conus geographus L. We have purified seven homologs of the mu-conotoxin set and determined their amino acid sequences, as follows, where Hyp = trans-4-hydroxyproline. GIIIA R.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro6]GIIIA R.D.C.C. T.P.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro7]GIIIA R.D.C.C.T.Hyp.P.K.K.C.K.D.R.Q.C.R.Hyp.Q.R.C.C.A-NH2 GIIIB R.D.C.C.T.Hyp.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2 [Pro6]GIIIB R.D.C.C.T.P.Hyp.R.K.C.K.D.R.R. C.K.Hyp.M.K.C.C.A-NH2 [Pro7]GIIIB R.D.C.C.T.Hyp.P.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2 GIIIC R.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.R.C.K.Hyp.L.K.C.C.A-NH2. Using the major peptide (GIIIA) in electrophysiological studies on nerve-muscle preparations and in single channel studies using planar lipid bilayers, we have established that the toxin blocks muscle sodium channels, while having no discernible effect on nerve or brain sodium channels. In bilayers the blocking kinetics of GIIIA were derived by statistical analysis of discrete transitions between blocked and unblocked states of batrachotoxin-activated sodium channels from rat muscle. The kinetics conform to a single-site, reversible binding equilibrium with a voltage-dependent binding constant. The measured value of the equilibrium KD for GIIIA is 100 nM at OmV, decreasing e-fold/34 mV of hyperpolarization. This voltage dependence of blocking is similar to that of tetrodotoxin and saxitoxin as measured by the same technique. The tissue specificity and kinetic characteristics suggest that the mu-conotoxins may serve as useful ligands to distinguish sodium channel subtypes in different tissues.     

Electrostatic and steric contributions to block of the skeletal muscle sodium channel by mu-conotoxin.

Pore-blocking toxins are valuable probes of ion channels that underlie electrical signaling. To be effective inhibitors, they must show high affinity and specificity and prevent ion conduction. The 22-residue sea snail peptide, mu-conotoxin GIIIA, blocks the skeletal muscle sodium channel completely. Partially blocking peptides, derived by making single or paired amino acid substitutions in mu-conotoxin GIIIA, allow a novel analysis of blocking mechanisms. Replacement of one critical residue (Arg-13) yielded peptides that only partially blocked single-channel current. These derivatives, and others with simultaneous substitution of a second residue, were used to elucidate the structural basis of the toxin's blocking action. The charge at residue-13 was the most striking determinant. A positive charge was necessary, though not sufficient, for complete block. Blocking efficacy increased with increasing residue-13 side chain size, regardless of charge, suggesting a steric contribution to inhibition. Charges grouped on one side of the toxin molecule at positions 2, 12, and 14 had a weaker influence, whereas residue-16, on the opposite face of the toxin, was more influential. Most directly interpreted, the data suggest that one side of the toxin is masked by close apposition to a binding surface on the pore, whereas the other side, bearing Lys-16, is exposed to an aqueous cavity accessible to entering ions. Strong charge-dependent effects emanate from this toxin surface. In the native toxin, Arg-13 probably presents a strategically placed electrostatic barrier rather than effecting a complete steric occlusion of the pore. This differs from other well-described channel inhibitors such as the charybdotoxin family of potassium channel blockers and the sodium channel-blocking guanidinium toxins (tetrodotoxin and saxitoxin), which appear to occlude the narrow part of the pore.     

Alpha 2-macroglobulin is a binding protein for basic fibroblast growth factor

After incubation with human serum or plasma, 125I-basic fibroblast growth factor (bFGF) (molecular mass 18.5 kDa) exhibits molecular mass forms greater than 200 kDa as determined by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. These high molecular mass forms of bFGF are immunoprecipitable with antiserum raised against alpha 2-macroglobulin (alpha 2M). Purified alpha 2M and 125I-bFGF form a covalent complex in a specific, saturable manner. Excess unlabeled bFGF competes with 125I-bFGF for complex formation. Complex formation is complete after 4 h and is inhibited by pretreating alpha 2M with dithiothreitol, iodoacetamide, iodoacetic acid, and N-ethylmaleimide. The complex is resistant to acidic conditions and denaturants such as urea. Heparin, which binds bFGF, has no effect on complex formation. Methylamine, which blocks protease binding to alpha 2M, increases the amount of 125I-bFGF that can be bound 2-fold. Plasmin and trypsin treatment of alpha 2M has no effect on 125I-bFGF binding. The ability of growth factors to compete for binding is specific, as aFGF and TGF-beta compete for binding to alpha 2M, whereas platelet-derived growth factor does not. 125I-bFGF.alpha 2M complexes do not bind to low affinity bFGF binding sites and bind poorly to high affinity bFGF binding sites on BHK-21 cells. In addition, 125I-bFGF bound to alpha 2M has decreased ability to stimulate plasminogen activator production in bovine capillary epithelial cells.     

Tertiary structure of conotoxin GIIIA in aqueous solution

The three-dimensional structure of conotoxin GIIIA, an important constituent of the venom from the marine hunting snail Conus geographus L., was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing based methods. On the basis of 162 assigned nuclear Overhauser effect (NOE) connectivities obtained at the medium field strength frequency of 400 MHz, 74 final distance constraints of sequential and tertiary ones were derived and used together with 18 torsion angle (phi, chi 1) constraints and 9 distance constraints derived from disulfide bridges. A total of 32 converged structures were obtained from 200 runs of calculations. The atomic root-mean-square (RMS) difference about the mean coordinate positions (excluding the terminal residues 1 and 22) is 0.8 A for backbone atoms (N, C alpha, C). Conotoxin GIIIA is characterized by a particular folding of the 22 amino acid peptidic chain, which is stabilized by three disulfide bridges arranged in cage at the center of a discoidal structure of approximately 20-A diameter. The seven cationic side chains of lysine and arginine residues project radially into the solvent and form potential sites of interaction with the skeletal muscle sodium channel for which the toxin is a strong inhibitor. The present results provide a molecular basis to elucidate the remarkable physiological properties of this neurotoxin.     

Molecular mechanisms of neurotoxin action on voltage-gated sodium channels

Voltage-gated sodium channels are the molecular targets for a broad range of neurotoxins that act at six or more distinct receptor sites on the channel protein. These toxins fall into three groups. Both hydrophilic low molecular mass toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Alkaloid toxins and related lipid-soluble toxins alter voltage-dependent gating of sodium channels via an allosteric mechanism through binding to intramembranous receptor sites. In contrast, polypeptide toxins alter channel gating by voltage sensor trapping through binding to extracellular receptor sites. The results of recent studies that define the receptor sites and mechanisms of action of these diverse toxins are reviewed here.     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Identification and partial characterization by chemical cross-linking of a binding protein for tissue-type plasminogen activator (t-PA) on rat hepatoma cells. A plasminogen activator inhibitor type 1-independent t-PA receptor.

Plasma tissue-type plasminogen activator (t-PA) is cleared rapidly in vivo by the liver. Previous studies with the human hepatoma cell line HepG2 have identified a clearance system for t-PA modulated by plasminogen activator inhibitor type 1 (PAI-1). In the present study, a rat hepatoma cell line MH1C1 is shown to contain a PAI-1-independent t-PA clearance system. At 4 degrees C, binding of 125I-t-PA to MH1C1 cells was rapid, specific, and saturable. Scatchard analysis of the binding data yielded a mean estimate of 105,000 high affinity binding sites per cell (Kd = 4.1 nM). When the bound ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the majority (about 90%) of the specific binding was in the form of uncomplexed 125I-t-PA. This is in contrast to HepG2 cells in which specific binding was mainly in the form of a sodium dodecyl sulfate-stable 125I-t-PA.PAI-1 complex. When availability of matrix-associated PAI-1 was blocked by preincubation with anti-PAI-1 antibody or removed by elastase treatment, specific 125I-t-PA binding to MH1C1 cells was unaffected, whereas most of the specific 125I-t-PA binding to HepG2 cells was abolished. Furthermore, when the active site of t-PA was inactivated with diisopropyl fluorophosphate, the diisopropyl fluorophosphate-t-PA specifically competed for binding of 125I-t-PA to MH1C1 cells, but failed to block specific 125I-t-PA binding to HepG2 cells. At 37 degrees C, PAI-1-independent t-PA binding to MH1C1 cells was followed by ligand uptake and degradation with kinetics similar to that seen in HepG2 cells. Chemical cross-linking of t-PA to MH1C1 cells revealed a specific t-PA binding protein with a molecular mass of about 500,000 daltons. Ligand-receptor complexes generated by chemical cross-linking were immunoprecipitable by anti-t-PA antibody but not by anti-PAI-1 antibody, further supporting the finding that binding of t-PA to MH1C1 cells is PAI-1-independent.     

Development of Sodium Channel Protein During Chemically Induced Differentiation of Neuroblastoma Cells

We have previously shown that the [3H]saxitoxin binding site of the sodium channel is expressed independently of the [125I]scorpion toxin binding site in chick muscle cultures and in rat brain. In the present work, we studied the development of the sodium channel protein during chemically induced differentiation of N1E-115 neuroblastoma cells, using [3H]saxitoxin binding, [125I]scorpion toxin binding, and 22Na uptake techniques. When grown in their normal culture medium, these cells are mostly undifferentiated, bind 90 +/- 10 fmol of [3H]saxitoxin/mg of protein and 112 +/- 14 fmol of [125I]scorpion toxin/mg protein, and, when stimulated with scorpion toxin and batrachotoxin, take up 70 +/- 5 nmol of 22Na/min/mg of protein. Cells treated with dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) differentiate morphologically within 3 days. At this time, the [3H]saxitoxin binding, the [125I]scorpion toxin binding, and the 22Na uptake values are not very different from those of undifferentiated cells. With subsequent time in DMSO or HMBA, these values continue to increase, a result indicating that the main period of sodium channel expression occurs well after the cells have assumed the morphologically differentiated state. The data indicate that the expression of sodium channels and morphological differentiation are independently regulated neuronal properties, that the attainment of morphological differentiation is necessary but not in itself sufficient for full expression of the sodium channel proteins, and that, in contrast to the chick muscle cultures and rat brain, the [3H]saxitoxin site and [125I]scorpion toxin site appear to be coregulated in N1E-115 cells.     

Effects of modification at the fifth residue of mu-conotoxin GIIIA with bulky tags on the electrically stimulated contraction of the rat diaphragm.

Mu-conotoxin GIIIA, a peptide toxin from the cone snail, blocks muscle-type sodium channels. Thr-5 of mu-conotoxin GIIIA, located on the opposite side of the active site in the globular molecule, was replaced by Cys to which the bulky tags were attached. The tagged mu-conotoxin GIIIA derivatives, except for the phospholipid-tagged one, exerted the biological activity with a potency slightly weaker than natural mu-conotoxin GIIIA. When the biotinylated tags of various lengths were added, the presence of avidin suppressed the action of the biotinylated toxins of <4 nm, but not with 5 nm. The bulky biotinylated tags are useful as a caliper to measure the depth of receptor sites in the channels.